而根據以往文獻的報導,IDS2p具有甲基轉移酶之特徵序列。我們將啤酒酵母菌(Saccharomyces cerevisiae)IDS2基因建構到大腸桿菌的Rosseta 菌株中作蛋白質表現,利用His-tag融合純化出所需要的IDS2p,並及將△IDS2菌株之蛋白質抽取液作受質,加入氚化甲基S-腺苷甲硫氨酸[C3H3]S-adenosy-L-methionine一起進行甲基化反應,接著將受質蛋白質加以分離。將受質蛋白質以直立式凝膠電泳isoelectric focusing (IEF)進行第一維的分離,然後將分離的受質蛋白質取下進行SDS-PAGE的第二維電泳,最後我們將分離出的受質蛋白質取下進行質譜儀的鑑定分析。 Proteins can be modified in several ways by the addition of methyl groups from S-adenosylmethionine. Methylation regulates a variety of biological functions including DNA and RNA metabolism, protein synthesis and signal transduction.
Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression. IDS2 (for IME2-dependent signaling) has a functional interaction with Ime2p.
According to recent studies, the sequence IDS2, is homologous to a methyltransferase. In our studies, we constructed the sequence IDS2 of Saccharomyces cerevisiae and overexpressed IDS2p in E. coli Rosetta (DE3) strains, and purified IDS2p with His-tag. The protein extract from △IDS2 strain was methylated with the cosubstrate [C3H3]S-adenosy-L-methionine prior to separation. The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel, and we cutted off the spot with labeled protein and separated the protein on SDS-PAGE. Finally the labeled protein was extracted from SDS-PAGE and identified by mass spectrometry.